Prostacyclin and prostaglandin synthesis in isolated brain capillaries

The synthesis of prostacyclin and prostaglandins was examined in isolated blood-free brain capillaries of guinea-pigs and rats using 1-14C-arachidonic acid as a precursor. The main prostaglandins synthesized by guinea-pig microvessels were prostaglandin D2 and prostaglandin E2. Substantially less prostaglandin F2 alpha or the prostacyclin stable metabolite, 6-oxo-prostaglandin F1 alpha was synthesized. Rat capillary prostaglandin distribution differed substantially from that of the guinea-pigs although the principle prostaglandin was also PGD2. Total prostaglandin conversion was greater in guinea-pig capillaries than in the rat. Norepinephrine stimulated the prostaglandin forming capacity of blood free cerebral microvasculature of guinea-pigs. Prostacyclin and prostaglandins could be involved in the activity dependent regulation of regional cerebral blood flow and permeability.     

Ketone body utilization for energy production and lipid synthesis in isolated rat brain capillaries

Isolated brain capillaries from 2-month-old rats were incubated for 2 h in the presence of [3-14C]acetoacetate, D-3-hydroxy[3-14C]butyrate, [U-14C]glucose, [1-14C]acetate or [1-14C]butyrate. Labelled CO2 was collected as an index of oxidative metabolism and incorporation of label precursors into lipids was determined. The rate of CO2 production from glucose was slightly higher than from the other substrates. Interestingly, acetoacetate was oxidized at nearly the same rate as glucose. This shows that ketone bodies could be used as a source of energy by brain capillaries. Radiolabelled substrates were also used for the synthesis of lipids, which was suppressed by the addition of albumin. The incorporation of [U-14C]glucose in total lipids was 10-times higher than that from other precursors. However, glucose labelled almost exclusively the glycerol backbone of phospholipids, especially of phosphatidylcholine. Ketone bodies as well as glucose were incorporated mainly into phospholipids, whereas acetate and butyrate were mainly incorporated into neutral lipids. The contribution to fatty acid synthesis of various substrates was in the following order: butyrate greater than or equal to acetate greater than ketone bodies greater than or equal to glucose. All precursors except glucose were used for sterol synthesis. Glucose produced almost exclusively the glycerol backbone of phospholipids.     

Sodium-dependent high-affinity uptake of taurine by isolated rat brain capillaries

Transport of taurine has been demonstrated in capillary preparations from adult rat brains using [3H]taurine. Taurine transport is mediated by a saturable high-affinity system which is entirely dependent on sodium ions. The apparent maximal influx (Vmax) and half-saturation concentration (Km) corresponded to 1.06.10(-4) mumol/min per mg protein and 27.5 microM, respectively. Competition experiments in the presence of sodium ion showed that [3H]taurine uptake was strongly inhibited by 0.1 mM unlabeled structural analogues of taurine such as beta-alanine and hypotaurine as well as unlabeled taurine. gamma-Aminobutyric acid (GABA) (0.1 mM) inhibited the uptake of labeled taurine by 30%, whereas isethionic acid, L-methionine, L-2,4-diaminobutyric acid, glycine, L-cysteinesulfonic acid and cystamine did not exhibit any inhibitory effect. The results suggest that the Na+ gradient is the principal source of energy for taurine transport into isolated brain capillaries. This transport system may play an active role in the regulation of taurine concentration in the brain extracellular space.     

Use of isolated brain capillaries and cultured endothelial cells to study the blood-brain barrier

Brain capillary endothelial cells are responsible for forming the blood-brain barrier (BBB). Methods are now available to isolate microvessels from brain and study their biochemical and transport characteristics. From these investigations, new ideas have been proposed concerning the role of endothelial cells in the function of the BBB. More recently, success in culturing endothelial cells from brain microvessels has opened the way for novel approaches to the study of the regulation of endothelial cell permeability. We anticipate continued rapid progress in this area and expect that this will lead to a better understanding of the mechanisms involved in the regulation of BBB permeability and brain capillary function.     

Stimulation of phospholipase A2 activity by oxygen-derived free radicals in isolated brain capillaries

An exogenous free radical generating system added to isolated brain capillaries induces degradation of phospholipids. This inductive effect reflects increased phospholipase activities as measured by fatty acid composition of various phospholipid fractions. The correlation of phospholipid degradation with stimulation of phospholipases was further investigated by using cationic amphiphilic agents, which are known to be phospholipase A2 inhibitors. The breakdown of phospholipids was inhibited by the pretreatment of isolated capillaries with these drugs.     

大鼠脑线粒体体外蛋白翻译体系的建立及蛋白产物的鉴定

目的 :建立大鼠脑组织线粒体的体外蛋白合成体系并对其合成产物进行电泳分离和分子量鉴定。方法 :分离大鼠脑组织线粒体 ,用3 H 亮氨酸掺入法探索线粒体体外翻译的最佳条件 ,3 5S 蛋氨酸掺入并对翻译后产物经SDS 聚丙烯酰胺凝胶电泳和放射自显影进行分子量鉴定。结果 :分离的线粒体氧化磷酸化偶联程度高 ,呼吸控制率(RCR)在 3.5～ 5 .5之间 ;体外3 H 亮氨酸的掺入活性在 6 0min内近似线性增长 ,而后维持在一相对稳定水平 ;3 H 亮氨酸的掺入活性随线粒体蛋白浓度而增加 ,而单位线粒体蛋白的掺入活性在 1mg/ml时最高 ;3 5S 蛋氨酸掺入SDS 聚丙烯酰胺凝胶电泳后可观察到清晰的 8条自显影带 ,分子量分别为 (单位Kda) 86、6 6、5 6、43、33、2 9、2 5、18。结论 :用此方法建立的脑线粒体离体翻译反应体系具有高活性和翻译忠实性等特点 ,是研究脑mtDNA在翻译水平的表达及调控的有效方法     

Acetoacetate,d-3-hydroxybutyrate and glucose utilization by capillaries isolated from developing rat brain

Isolated cerebral capillaries from developing rats utilize glucose as well as ketone bodies essentially for oxidative metabolism. However, CO₂ production from [U-¹⁴C]glucose was significantly greater than from ketone bodies (except at 5 mM). Ketone body utilization (in the presence of 5 mM glucose in the incubation medium) was concentration-dependent (up to 5 mM). Lipid synthesis from ketone bodies was comparable to that from glucose up to 1 mM. At concentrations ⩾ 1 mM, acetoacetate incorporation into total lipids and fatty acids was higher than other substrates, however, this difference was statistically significant only at 5 mM. Incorporation of substrates into sterols was very low (> 1 pmol/h/mg protein).     

Phenylalanine transport at the human blood-brain barrier. Studies with isolated human brain capillaries

The exquisite sensitivity of brain amino acid availability to changes in plasma amino acid composition arises from the uniquely high affinity (low Km) of blood-brain barrier transport sites as compared to cell membrane transport systems in nonbrain tissues. The extension of this paradigm from rats to man assumes that the Km of blood-brain barrier amino acid transport in the human is low as in the rat. This hypothesis is tested in the present studies wherein isolated human brain capillaries are used as a model system for the human blood-brain barrier. Capillaries were obtained from autopsy brain between 20 and 45 h after death and were isolated in high yield and free of adjoining brain tissue. [3H]Phenylalanine transport into the isolated human, rabbit, or rat brain capillary was characterized by two saturable transport systems and a nonsaturable component. The Km values of phenylalanine transport into brain capillaries via the two saturable systems averaged 0.26 +/- 0.08 and 22.3 +/- 7.1 microM for five human subjects. These studies provide the first evidence for a very high affinity (Km = 0.26 microM) neutral amino acid transport system at the blood-brain barrier, and it is hypothesized that this system is selectively localized to the brain side of the blood-brain barrier. The results also show that the transport Km values for phenylalanine transport are virtually identical at both the rat and human blood-brain barrier.     

Prostacyclin and thromboxane synthesis by endometrial cancer and leiomyomas

To study the role of prostacyclin (PGI₂) and thromboxane A₂ (TxA₂) in uterine tumors, pieces of endometrial cancer (n=12) and leiomyomas (n=12)_were incubated in vitro, and the productions of 6-keto-prostaglandin F_1a (6-keto-PGF_1a, a hydration product of PGI₂) and thromboxane B₂ (TxB₂, a hydration product of TxA₂), measured by radioimmunoassay, were compared to those of corresponding healthy tissues. The production of 6-keto-PGF_1a by endometrial cancer (20.8; 1.5–85 ng/mg protein/min, median and interquartile range), by healthy endometrium (25.5; 10.0–55.0), by healthy myometrium (34.9; 25.0–59.9) and by leiomyoma (20.3; 10.2–45.1) was similar. The production of TxB₂ was increased by endometrial cancer (55.5; 10.5–155.2, p < 0.02) in comparison with endometrium (9.8; 4.3–35.1), myometrium (3.8; 2.1–8.0) and leiomyoma (1.9; 1.0–3.8). The 6-keto-PGF_1a/TxB₂ ratio in endometrial cancer (0.9; 0.3–1.5) was smaller (p < 0.02) than that in healthy endometrium (3.3; 1.9–4.8). Thus, TxA₂ may be a factor in endometrial cancer.     

Prostacyclin and thromboxane synthesis by endometrial cancer and leiomyomas

To study the role of prostacyclin (PGI2) and thromboxane A2 (TxA2) in uterine tumors, pieces of endometrial cancer (n = 12) and leiomyomas (n = 12) were incubated in vitro, and the productions of 6-keto-prostaglandin F1a (6-keto-PGF1a, a hydration product of PGI2) and thromboxane B2 (TxB2, a hydration product of TxA2), measured by radioimmunoassay, were compared to those of corresponding healthy tissues. The production of 6-keto-PGF1a by endometrial cancer (20.8; 15.1-85.0 ng/mg protein/min, median and interquartile range), by healthy endometrium (25.5; 10.0-55.0), by healthy myometrium (34.9; 25.0-59.9) and by leiomyoma (20.3; 10.2-45.1) was similar. The production of TxB2 was increased by endometrial cancer (55.5; 10.5-155.2, p less than 0.02) in comparison with endometrium (9.8; 4.3-35.1), myometrium (3.8; 2.1-8.0) and leiomyoma (1.9; 1.0-3.8). The 6-keto-PGF1a/TxB2 ratio in endometrial cancer (0.9; 0.3-1.5) was smaller (p less than 0.02) than that in healthy endometrium (3.3; 1.9-4.8). Thus, TxA2 may be a factor in endometrial cancer.     

Increased brain capillaries in chronic hypoxia.

The effect of chronic hypobaric hypoxia (28 days, 455 Torr) on the organization of brain vessels was studied in Balb/c mice. In comparison to age-matched controls kept at sea level, emulsion-perfused capillaries in hypoxic mice showed marked dilation in all brain areas studied. Capillary length per unit volume of tissue (Lv) was increased in the cerebellar granular layer, the caudate nucleus, the globus pallidus, the substantia nigra, the superior colliculus, and the dentate gyrus. There was a selective increase of Lv in the hippocampus (CA1 strata pyramidale and lacunosum and CA3 strata pyramidale and oriens) and in somatosensory cortex layers V and VI, motor cortex layers II, III, V, and VI, and auditory cortex layers II and III. An increase in capillary surface area per unit volume of tissue was also determined in several brain areas, including layer IV of somatosensory cortex, where Lv was not significantly increased. The O2 diffusion conductance and PO2 in the tissues were estimated with a mathematical model. The remodeling of capillary diameter and length during chronic hypoxia accounts for the significant increase of O2 conductance to neural tissues. Also the estimated tissue PO2 in chronic brain hypoxia is markedly increased in the caudate nucleus and the substantia nigra compared with acute hypoxia. These results suggest that formation of new capillaries is an important mechanism to restore the O2 deficit in chronic brain hypoxia and that local rates of energy utilization may influence angiogenesis in different areas of the brain.     

On the ability of galactose to influence hexose and amino acid uptake by isolated rat brain capillaries

The uptake of glucose, 2-deoxyglucose, proline, methionine, and alpha-methylamino isobutyric acid was studied in brain capillaries preincubated with 50 mM galactose either in vitro or isolated from galactose-fed rats. The uptake was not decreased in both cases. The linear rate of glucose oxidation by capillaries was also not altered by preincubation with galactose.     

Choline and ethanolamine glycerophospholipid synthesis in isolated synaptosomes of rat brain.

Substantial activities of cholinephosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) were found with lysed synaptosomes but not with intact synaptosomes isolated from adult rat brains. Synaptosomal and non-synaptosomal microsomal transferases were similar in kinetic properties. Substantial activities of synaptosomal transferases have not been described previously. Part of the glycerophospholipids in synaptosomal membranes may be synthesized in the nerve ending in addition to the glycerophospholipids supplied by axonal transport. The synthesis of the alkylacyl type of choline and ethanolamine glycerophospholipids was moderately inhibited by 1 mM ATP and 1 microM cyclic AMP. This synthesis was also inhibited by more than 50% by 1 mM norepinephrine and to a lesser extent by 5 mM hydroxytryptamine and 1 mM acetylcholine. Cyclic AMP may mediate the effects of biogenic amines. The relative synthesis of different glycerophospholipid classes and the relative proportion of alkylacyl type (plasmalogen precursors) and diacyl type of glycerophospholipids may be influenced by the levels of adenine nucleotides and/or biogenic amines. Elevated cyclic AMP levels will decrease the synthesis of plasmalogen precursors.